Rajković, J., Joković, N.  Probiotic properties and safety assesment… BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 81 Original Article Received: 02 December 2015 Revised: 14 December 2015 Accepted: 20 December 2015 Probiotic properties and safety assessment of lactic acid bacteria isolated from kajmak Jelena Rajković, Nataša Jokovića* University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Višegradska 33, 18000 Niš, Serbia * E-mail: jole35@mts.rs Abstract: Rajković, J., Joković, N.: Probiotic properties and safety assessment of lactic acid bacteria isolated from kajmak. Biologica Nyssana, 6 (2), December 2015: 81-89. One hundred forty-one strains of lactic acid bacteria (LAB) were tested for probiotic features such as antimicrobial activities, autoaggregation ability, hydrophobicity of bacterial cell walls and ability to grow on and hydrolyze bile salts. Additionally, resistance to antibiotics and production of biogenic amines were performed as safety assessment of LAB strains. Five Lc. lactis ssp. lactis and six Lc. raffinolactis strains showed antibacterial activities against used indicator strains. Protein nature of antibacterial compounds was proven by the addition of pronase. Enterococcus durans strains had the highest degree of autoaggregation and hydrophobicity while the rest of the strains showed low degree for both characteristics. Lb. paracasei, Lb. plantarum, Ln. mesenteroides, En. faecium, En. faecalis and En. durans strains could growth on bile salts and some of them were able to hydrolyse them. Safety assessment of LAB indicates that many strains of enterococci were resistant to chloramphenicol, tetracycline, cephalosporin and to the lowest used concentration of erythromycin. The strains from other LAB genera were resistant to antibiotics used in lower concentration and in smaller numbers when compared to enterococci. Key words: Lactic acid bacteria, Probiotic properties, Safety assessment Apstrakt: Rajković, J., Joković, N.: Probiotska svojstva i bezbednost upotrebe mlečno kiselinskih bakterija izolovanih i kajmaka. Biologica Nyssana, 6 (2), December 2015: 81-89. Probiotska svojstva mlečno kiselinskih bakterija kao što su antimikrobna aktivnost, autoagregacija, hidrofobnost i sposobnost da rastu na žučnim solima i vrše njihovu hidrolizu ispitivana su na sto četrdeset jednom bakterijskom izolatu. Pored toga, ispitivana je rezistentnost ovih bakterijih izolata na antibiotike i sposobnost produkcije biogenih amina u okviru procene bezbednosti izolata za upotrebu u starter kulturama. Pet Lc. lactis ssp. lactis i šest Lc. raffinolactis izolata pokazala su antimikrobno delovanje prema korišćenim indikatorskim sojevima. Proteinska priroda antimikrobnih jedinjenja je potvrdjena dodavanjem pronaze. Najveći stepen hidrofobnosti i autoagregacije imali su En. durans izolati, dok su kod ostalih izolata ova probiotska svojstava bila izražena u manjem stepenu. Lb. paracasei, Lb. plantarum, Ln. mesenteroides, En. faecium, En. faecalis i En. durans izolati su pokazali rast na podlogama sa žučnim solima, dok su neki od njih imali sposobnost da vrše njihovu hidrolizu. Procenom sigurnosti upotrebe izolata utvrđeno je da su mnogi izolati enterokoka bili rezistentni na hloramfenikol, tetraciklin, cefalosporin i na najniže korišćene koncentracije eritromicina. Izolati iz ostalih rodova mlečno kiselinskih bakterija su bili rezistentni na niske koncentracije antibiotika, ali u manjem broju u poređenju sa enterokokama. Key words: mlečno kiselinske bakterije, probiotska svojstva, procena bezbednosti 6 (2) • December 2015: 81-89 BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 82 Introduction Lactic acid bacteria (LAB) are a heterogeneous group of bacteria, including Gram-positive cocci and bacilli, with common feature to produce lactic acid as the end-product of metabolism. They are mainly isolated from the various fermented foods produced from milk, meat or vegetables but also present a normal microflora of the nasopharyngеal, intestinal and vaginal mucosa of humans and animals (S t i l e s & H o l z a p f e l , 1997). Various strains of LAB are widely used in the industry as a part of starter cultures for improving the quality and shelf life of fermented foods in different food processes. In recent years, LAB has been intensively studied as probiotic bacteria. Probiotics are defined as "live microorganisms which when administered in adequate amounts confer a health benefit to the host'' (F A O / W H O , 2002). In dairy products bifidobacteria and lactobacilli are commonly used as probiotic bacteria, but other LAB, such as enterococci, lactococci or streptococci, also may possess probiotic properties (P r i n g s u l a k a et al., 2015). Intake of probiotic bacteria could lead to the establishment of intestinal microbial balance, prevention of allergies and alleviation of certain intolerances. Antimutagenic, anticarcinogenic, hypocholesterolemic, antihypertensive, anti- osteoporosis, and immunomodulatory effects on the host have been also reported for some probiotic bacteria (C h i a n g & P a n , 2012). Considering the fact that beneficial effects of LAB are strain-dependent, screening of LAB isolated from different sources for probiotic properties is a common way for searching new probiotic bacteria. Synthesis of antimicrobial compounds, adhesion potential to the intestinal epithelium and ability to survive conditions in the gastrointestinal tract are the characteristics that have been studied for preliminary selection of potential probiotics (B a u t i s t a - G a l l e g o et al., 2013; P r i n g s u l a k a et al., 2015; A n g m o et al., 2016). The strains of LAB which have capacity to produce different antimicrobial compounds could inhibit the pathogenic bacteria in gastrointestinal tract, thus reducing the risk of gastrointestinal diseases. In order to achieve the effect in intestine, probiotic bacteria must have ability to pass through upper parts of gastrointestinal tract and adhere to intestinal epithelial cells. Conditions in the upper gastrointestinal tract, such as pH values and bile salts, may adversely affect the probiotic bacteria. Therefore, it is necessary to examine the possibility of survival of the probiotic bacteria in the presence of bile salts. Aggregation ability and hydrophobicity of bacterial cell walls are correlated to adherence properties of probiotic bacteria (D u a r y et al., 2011; J a n k o v i ć et al., 2012). Required property of potential probiotic bacteria is the safety for human consumption. Resistance to different antibiotics, the presence of virulence factors and synthesis of biogenic amines are commonly tested undesirable characteristics of probiotic bacteria (C h a m b a & J a m e t , 2008). Traditional fermented products are the source of the new probiotic bacteria that can be used in the production of various types of functional foods. The aim of current study was screening of LAB isolated from Serbian traditional dairy product, kajmak, for some probiotics features such as antimicrobial activities, autoaggregation ability, hydrophobicity of bacterial cell walls and the ability to grow on and to hydrolyze bile salts. Additionally, LAB strains were tested for resistance to antibiotics and production of biogenic amines as safety assessment of strains. Material and methods Bacterial strains, media, and growth conditions One hundred forty-one strains of LAB isolated from different samples of kajmak were used in this study. All strains were previously identified by molecular methods to the species level as Lactococcus lactis ssp. lactis (19), Lactococcus raffinolactis (6), Streptococcus thermophilus (4), Enterococcus faecalis (9), Enterococcus faecium (19), Enterococcus durans (10), Leuconostoc mesenteroides (50), Lactobacillus plantarum (15), Lactobacillus paracasei (6), Lactobacillus satsumensis (1), Lactobacillus kefiri (1) and Lactobacillus kefiranofaciens (1) (J o k o v i c et al., 2008; J o ko vi ć et al., 2014). The strains were stored at -20 C and activated by triple streaking on appropriate media. MRS agar (Merck, GmbH, Darmstadt, Germany) was used for growth of Leuconostoc and Lactobacillus strains whereas Lactococcus, Streptococcus and Enterococcus strains were cultured on M17 agar (Merck, GmbH, Darmstadt, Germany) supplemented with glucose (0.5%, w/v; GM17 broth). The inoculated media were incubated overnight at 30 C for mesophilic and 37 C for thermophilic strains. Tryptic soy agar (Himedia, India) was used for the growth of non-LAB strains (Escherichia coli, Bacillus subtilis and Listeria innocua) which were incubated at 37 C for 24 h. Yeast (Candida albicans) was cultured on Sabouraud maltose agar (Torlak, Serbia) at 30 C for 48 h. BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 83 Antimicrobial activity of strains The LAB strains were screened for antimicrobial activity by the agar-well diffusion method (T a g g & M c G i v e n , 1971) using various indicator strains that are listed in Table 1. Asssay was performed by overlaying solid MRS or GM17 medium in Petri dishes with 5 ml of soft (0.7% agar) MRS, GM17, Tryptic soy and Sabouraud maltose media inoculated with 105-106 cells of indicator strain/ml medium. The wells, 5 mm in diameter, were made in soft agar and filled with 100 l of overnight strain culture with a potential of bacteriocin production. The presence of antimicrobial substances was detected by the appearance of clear zones around wells as a result of growth inhibition of sensitive bacterial strains. The protein nature of bacteriocins was confirmed by adding a cristal of pronase E near to the edge of the well. Absence of a zone of inhibition in the place where pronase E crystals were added indicates the protein nature of bacteriocins. Adhesive properties of strains Bacterial adhesion to hydrocarbons was determined by the method described by F o r t i n a et al. (2008). Cells from overnight culture were struck down by centrifugation at 5000 rpm for 15 min, washed twice in PBS buffer and resuspended in 0.1 M KNO3 (pH 6.2). The initial absorbance of the cell suspension was adjusted to 0.5-0.6 at 600 nm (A0). Then, 3 ml of cell suspension was mixed with 1 ml of n- hexadecane. The mixture was incubated at room temperature for 10 min and mixed well on vortex for 2 min. After 20 min of incubation at room temperature, the aqueous phase was carefully separated and the optical density was measured at 600 nm (At). The percentage of bacterial adhesion to n-hexadecane was expressed as: % hydrophobicity = 1- (At/A0) x100, where A0 and At represent absorbance values of aqueous phase before and after contact with n-hexadecane. Aggregation of strains was monitored in phosphate buffered saline (PBS) according to F o r t i n a et al. (2008). Bacterial cells from overnight cultures were centrifuged at 5000 rpm for 15 min, washed twice in PBS and resuspended in 4.0 mL of PBS to give an OD600nm of 1.0. The cell suspensions were further well mixed on a vortex for 10 s and autoaggregation was determined during 5 h of incubation at room temperature. Every hour, 100 l from the surface of the suspension was transferred to new microtube containing 900 l PBS buffer and OD600nm was measured. The percentage of aggregated cells was calculated as 1-(At/A0) x100 where A0 represents absorbance values at time t=0, while At represents absorbance values at time t= 5 h. The growth of strains on bile salts The growth of strains in the presence of bile salts was monitored on LAPTg medium consisted of: 15 g/l peptone, 10 g/l tryptone, 10 g/l yeast extract, 10 g/l glucose, 15 g/l agar, 0.01 ml/l of tween 80 and 0.3 g/l bovine bile (F o r t i n a et al., 2008). The ability of strains to hydrolyse bile salts was detected by precipitation of deconjugated bile acids around the colony that grew on the LAPTg medium without bovine bile and with the addition of 0.05 g/l bile salts No. 3 (Torlak, Belgrade, Serbia) and 0.2 g/l CaCl2. A positive reaction for the presence of hydrolase is the appearance of a precipitate around the colonies. Safety assessment The resistance of strains to antibiotics was tested by spot agar test on antibiotic-containing GM17 and MRS agar media optimal for LAB growth. Tests were done for the following antibiotics and concentration: chloramphenicol (10 g/ml, 20 g/ml), tetracycline (10 g/ml, 20 g/ml, 30 g/ml) and erythromycin (5 g/ml, 10 g/ml). Table 1. List of strains used in antimicrobial activity assay Bacterial strains Source or reference Lactococcus lactis ssp. lactis biovar. Diacetylactis, S50 (Kojic et al., 2005) Lactococcus lactis ssp.cremoris, NS1 (Kojic et al., 1991) Lactococcus lactis ssp. Lactis, BGMN1-596 (Gajic et al., 1999) Lactococcus lactis ssp. Lactis, NP45 Lab. kolekcija Lactobacillus paracasei ssp. Paracasei, BGBUK2-16/K4 (Lozo et al., 2004) Lactobacillus plantarum, A112 (Vujcic & Topisirovic, 1993) Escherichia coli, ATCC 25923 ATCC Bacilus subtilis, ATCC 6633 ATCC Listeria innocua, ATCC 33090 ATCC Candida albicans, ATCC 10231 ATCC BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 84 The ability of LAB isolates to produce biogenic amines was determined according to the method described by B o v e r -C i d and H o l z a p f e l (1999). Histidine, tyrosine, ornithine and lysine were used as precursors for the biogenic amines synthesis. The color change of colonies to purple or occurrence of precipitates around the colonies in case of tyramine synthesis indicated the decarboxylation activities of isolates. Results and discussion Antimicrobial activity LAB produce different substances that have antimicrobial activity, such as an organic acid (lactic, formic, acetic acid etc.), CO2, H2O2, diacetyl, ethanol, and bacteriocins. Among these products, bacteriocins are the most important because of their possible application in the food industry as biopreservatives (D e V u y s t & L e r o y , 2007). Since bacteriocins have the highest inhibitory effect against related Gram positive bacteria, bacteria from genus Lactococcus, Lactobacillus, Bacillus and Listeria were used as indicator strains (Tab. 1). Additionally, Gram negative bacteria E. coli and yeast C. albicans were also used in antimicrobial assays. Results of antimicrobial assays showed that a small number of strains could synthesize bacteriocins (Tab. 2). Five Lc. lactis ssp. lactis, and six Lc. raffinolactis strains produced bacteriocins with inhibitory effect on the growth of lactococcal indicator strains Lc. lactis ssp. cremoris NS1, Lc. lactis ssp. lactis BGMN1-596 and Lc. lactis ssp. lactis NP45 (Tab. 2). Lactococcal strains formed Table 2. Antimicrobial effect of tested strains shown through the zones of inhibition Species N o . o f is o la te s Indicator strains Lactococcus lactis ssp. lactis BGMN1-596 Lactococcusl actis ssp. cremoris NS1 Lactococcus lactis ssp. lactis NP45 Lactococcus lactis ssp. lactis biovar. diacetylactis S50 Escherichia coli ATCC2592 Listeria innocua ATCC 33090 Lc. lactis ssp. lactis 3 1 mm S 1 mm S 1 mm S - - - Lc. lactis ssp. lactis biovar. diacetylactis 4 7 mm S 7 mm S 3 mm S - - - Lc. raffinolactis 6 5 mm S 5 mm S 3 mm S 2 mm S 1 mm S 2 mm S En. faecium 1 - - - 2 mm S 5 mm S En. faecalis 1 4 mm S 2 mm S - - - 0.5 mm S mm – zone width; S – clear zone. Table 3. Autoaggregation, hydrophobicity, growth on bile salts and hydrolysis of bile salts of tested LAB strains L c . la c ti s s sp . la c ti s (1 9 )1 L c . ra ff in o la c ti s (6 ) S t. t h e rm o p h il u s (4 ) E n . fa e c a li s (9 ) E n . fa e c iu m ( 1 9 ) E n . d u ra n s (1 0 ) L n . m e se n te ro id e s (5 0 ) L b . p a rc a se i ( 6 ) L b . p la n ta ru m ( 1 5 ) L b . sa ts u m e n si s (1 ) L b . k e fi ri ( 1 ) L b . k e fi ra n o fa c ie n s (1 ) Hydrophobicity (%)2 19 2 10 14 28 45 17 6 6 27 2 3 Autoaggregation (%)2 39 31 36 31 44 71 36 50 27 36 8 37 Growth in the presence of bile salts3 - - - 4 10 10 3 3 9 - - - Hydrolysis of bile salts 3 - - - 1 4 5 3 3 8 - - - 1 -numbers in parenthesis represent the number of isolates 2 -results are presented as mean value 3 -results are shown as the number of isolates that has a certain property BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 85 Fig. 1. Inhibitory effects of Lc. lactis ssp. lactis and Lc. raffinolactis strains on Lc. lactis ssp. cremoris NS1 as indicator strain clear zones of inhibition, which lacked on the points where pronase E was added (Fig. 1). Lc. raffinolactis strains also inhibited the growth of Lc. lactis ssp. lactis biovar. diacetylactis S50, E. coli ATCC 2592 and L. inoccua ATCC 33090 but zones of inhibition were smaller in these cases (Tab. 2). One strain of En. faecium formed clear zones of inhibition against Lc. lactis ssp. lactis biovar. diacetylactis S50, and L. innocua ATCC 33090 while En. faecalis strain had inhibitory effects on Lc. lactis ssp. cremoris NS1, Lc. lactis ssp. lactis BGMN1-596 and L. inoccua ATCC 33090 (Tab. 2). Zones of inhibition formed by enterococci were not sensitive to pronase E, so it can be concluded that the antimicrobial substance doesn’t have protein nature. Strains which have been found to have antimicrobial activity against any of the above mention indicator strains, did not act inhibitory against Lb. plantarum A112, Lb. paracasei ssp. paracasei BGBUK2-16 / K4, B. subtilis ATCC 6633 and C. albicans ATCC 10231 indicator strains. Synthesis of bacteriocins is significant characteristics of probiotic bacteria because bacteriocin producing strains could inhibit pathogenic bacteria in the gastrointestinal tract. Except probiotic effects that strains with antimicrobial activities could have, bacteriocin producing LAB strains are a part of starter cultures for improving the microbiological quality and safety of dairy products, or for accelerating the ripening of fermented foods (A y a d et al., 2004; M o h a m m e d et al., 2009). Adhesive properties of strains Autoaggregation and hydrophobicity, as surface characteristics of the LAB strains, indicate potential adhesion ability of probiotic LAB strains to epithelial cells of human intestine (F o r t i n a et al., 2008). The high degree of autoaggregation indicates better adhesive capability of isolates, while physicochemical properties of the cell wall of LAB may probably affect autoaggregation and adhesion (F o r t i n a et al., 2008). According to M a l d o n a d o et al. (2012), three different groups of LAB strains can be distinguished on the basis of autoaggregation and hydrophobicity degrees: high (60–100%), medium (30–60%) and low (0–30%). The average values of these characteristics of tested LAB species are summarized in Table 3. Enterococcus durans strains showed the highest autoaggregation and hydrophobicity degrees with the average values of 71% and 45%, respectively. Among six En. durans strains, three of them aggregated quickly after 15 min, forming a precipitate and clear solution. These strains also expressed hydrophobicity values higher than 60%. Lc. lactis, Lc. raffinolactis, St. thermophilus, En. faecalis, En. faecium, Ln. mesenteroides, Lb. paracasei, Lb. satsumensis and Lb. kefiranofaciens strains showed a medium degree of autoaggregation. En. faecium and Lb. paracasei strains had the highest degree of autoaggregation in this group (44% and 50%, respectively). All strains in this group had very low level of binding to n-hexadecane and only En. faecium strains had average degree of hydrophobicity higher than 20%. Lb. plantarum strains had low degree of autoaggregation and hydrophobicity (27% and 6%, respectively) while one Lb. kefiranofaciens strain had the lowest degree of both properties among tested strains. High intraspecies variability in autoaggregation and hydrophobicity degrees was observed for LAB strains evidencing that these properties seems to be strain specific. Similar findings have been reported by other authors (T o d o r o v et al., 2008; G i a o u r i s et al., 2009; I y e r et al., 2010). Growth in the presence of bile salts The growth of the LAB strains in the presence of bile salts is a significant characteristic of probiotic LAB strains, which indicates the possibility of their survival in gastrointestinal tract (D u n n e et al., 2001). BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 86 Table 4. Resistance of LAB strains to different antibiotics Antibiotic Chloramphenicol, g/ml Tetracycline,g/ml Erythromycin, g/ml Cephalosporin,g/ml LAB species 10 20 10 20 30 5 10 10 20 30 Lc. lactis ssp. lactis (19)1,2 5 - 19 18 - 8 - 19 18 18 Lc. raffinolactis (6) - - - - - - - - - - St. thermophilus (4) - - 4 2 1 - - 4 2 2 En. faecalis (9) 9 6 9 9 3 8 - 9 9 9 En. faecium (19) 19 13 19 19 5 19 - 19 19 19 En. durans (10) 8 - 10 10 7 - - 10 10 10 Ln. mesenteroides(50) - - 50 45 4 40 - 50 40 9 Lb. parcasei (6) - - 6 6 - 4 - 6 6 4 Lb. plantarum (15) - - 15 14 - 14 4 15 14 12 Lb. satsumensis (1) - - 1 1 - 1 - 1 1 1 Lb. kefiri (1) - - 1 1 - - - 1 1 1 Lb. kefiranofaciens (1) - - - - - - - - - - 1 -numbers in parenthesis represent the number of isolates of the certain strain 2 -the results are presented as number of isolates which are resistant to a particular antibiotic The ability of bile salts hydrolysis is desirable probiotic characteristics of LAB strains, because the bile salts deconjugation contributes to the intestinal microflora balance (M a t h a r a et al., 2008). Selection of probiotic strains based on their deconjugation activities is also important, because of the correlation between this activity and the ability of LAB strains to decrease cholesterol level (P e r e i r a et al., 2003). In our study, both characteristics were detected only in strains of genus Enterococcus, Leuconostoc and Lactobacillus (Tab. 3). The most of the strains that could grow in the presence of bile salts belonged to species En. faecium, En. durans and Lb. plantarum while half of the Lb. plantarum strains could hydrolyse bile salts. The growth of Lb. plantarum, Lb. paracasei and En. faecium strains on the media with the addition of bile salts was also found in earlier studies (X a n t h o p o u l o s et al., 2000; S a a v e d r a et al., 2003; T o d o r o v et al., 2008). Certain Lb. plantarum, Lb. paracasei and En. faecium strains were found to be able to deconjugate bile salts (S a a v e d r a et al., 2003; M a r a g k o u d a k i s et al., 2006; N g u y e n et al., 2007). Safety assessment Although LAB has GRAS (generally recognized as safe) status, the strains isolated from fermented foods that can be used as starter culture should be tested for their potentially adverse effects on human health. Antibiotic resistance and the synthesis of biogenic amines are mostly analyzed characteristics of LAB strains important for their safe use in starter cultures (F r a n c i o s i et al., 2009). Antibiotic resistance of LAB strains is a major problem in the food industry after the discovery that certain strains of lactococci, lactobacilli and enterococci have genes for antibiotics resistance located on plasmids and transposons. Thus, antibiotic resistance can be transferred to other bacterial species (K a s t n e r et al., 2006). In our work, resistance to antibiotics was determined by growing the isolates on the appropriate medium with various antibiotics (chloramphenicol, tetracycline, erythromycin, and cephalosporin) added in certain concentrations (Tab. 4). Cephalosporin belongs to the group of antibiotics which inhibit the synthesis of the cell wall, while tetracycline, erythromycin, chloramphenicol affect the protein synthesis in the bacterial cell. Results from the present study showed that the most of the tested LAB strains were resistant to cephalosporin (Tab. 4). All enterococci, Lb. satsumensis strain, Lb. kefiri strain and the most of Lc. lactis ssp. lactis, St. thermophilus, Lb. paracasei and Lb. plantarum strains grew well on the media where cephalosporin was added in the concentration of 30 µg/ml. Strains belonging to species Ln. mesenteroides showed the highest sensitivity to cephalosporin with only 9 strains resistant to the highest concentration of cephalosporin used (Tab. 4). BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 87 The most of the LAB strains were resistant to tetracycline when it was added in media in low concentration (10 g/ml and 20 g/ml). Increasing of tetracycline concentration in media reduced the number of resistant strains for all LAB species but some strains of enterococci were resistant to this high concentration of tetracycline. When chloramphenicol was added in the concentration of 10 g/ml, all enterococcal strains and a small number of lactococcal strains were resistant. Increasing concentrations of chloramphenicol at 30 g/ml did not affect growth of the most of En. faecium and En. faecalis strains. Addition of erythromycin in a concentration of 5 µg/ml in media did not inhibit the growth of En. faecium, En. faecalis, Ln. mesenteroides, Lb. paracasei, Lb. plantarum and Lb. satsumensis strains, but some of Lc. lactis, all St. thermophilus and En. durans strains and one Lb. kefiri strain were sensitive to addition of this antibiotic in low concentration. Only four Lb. plantarum strains were resistant to the higher concentration of erythromycin (10 µg/ml). The most sensitive to the tested antibiotics were Lc. raffinolactis and Lb. kefiranofaciens strains that showed no growth on any of the medium with the addition of antibiotics. Antibiotic resistant strains from dairy environment were also found by other researchers (F l ó r e z et al., 2005; D e v i r g i l i i s et al., 2010). Data on proposed minimum inhibitory concentrations of antibiotics used for LAB isolates from dairy products are different in the literature due to the differences between selected methods (A m m o r et al., 2007). Overall, LAB isolates from dairy products are the most sensitive to erythromycin, while their resistance is most pronounced to cephalosporins (K a t l a et al., 2001; T e m m e r m a n et al., 2003). Resistance to chloramphenicol and tetracycline is distinguished among LAB isolates from different genus and it is usual to obtain strains with a much higher resistance compared to the proposed minimal inhibitory concentration for a particular species (D a n i e l s e n & W i n d , 2003; F l ó r e z et al., 2005; M a r a g k o u d a k i s et al., 2006). In fermented food, biogenic amines formed by bacterial decarboxylation of amino acids histidine, tyrosine, lysine or ornithine, lead to the undesirable flavor of final product and may have negative impact on human health (L a n d e t e et al., 2007). Among analyzed LAB strains, all En. faecalis strains, 12 strains of En. faecium and 5 strains of En. durans synthesized tyramine from tyrosine. Production of tyramine is commonly detected characteristics of enterococci from dairy products (M a r t í n - P l a t e r o et al., 2009). Conclusion Traditional fermented products, such kajmak is, are new sources of LAB strains with interesting characteristics. In recent years, LAB strains that have probiotic properties are particularly popular because they can be used for the production of new types of functional foods. The results of the present research indicate that among analyzed LAB strains isolated from kajmak, a few strains had interesting probiotic characteristics. Five Lc. lactis ssp. lactis and six Lc. raffinolactis strains synthesized bacteriocins, so they could be used for a control of pathogenic bacteria. Enterococcus durans strains with high degree of autoaggregation and hydrophobicity should be further tested for adhesion to human cell lines. Strains of Lb. paracasei, Lb. plantarum and Ln. mesenteroides that hydrolyze bile salts could have cholesterol lowering effects. All the above- mentioned strains were low resistance to used antibiotics, and they didn’t have the ability to synthesize biogenic amines. On the other hand, all enterococcal strains that hydrolyze bile salts showed high resistance to used antibiotics and could decarboxylate amino acids. Acknowledgements. The authors are grateful to the Ministry of Education, Science and Technological Development for financial support through the grant within frame of basic research, No TR 31032. References Ammor, MS., Flórez, AB., Mayo, B. 2007: Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria. Food Microbiology, 24 (6): 559-570. Angmo, K., Kumari, A., Bhalla, TC. 2016: Probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of Ladakh. LWT-Food Science and Technology, 66: 428-435. Ayad, E., Nashat, S., El-Sadek, N., Metwaly, H., El- Soda, M. 2004: Selection of wild lactic acid bacteria isolated from traditional Egyptian dairy products according to production and technological criteria. Food Microbiology, 21 (6): 715-725. Bautista-Gallego, J., Arroyo-López, F., Rantsiou, K., Jiménez-Díaz, R., Garrido-Fernández, A., Cocolin, L. 2013: Screening of lactic acid bacteria isolated from fermented table olives with probiotic potential. Food Research International, 50 (1): 135-142. BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 88 Bover-Cid, S., Holzapfel, WH. 1999: Improved screening procedure for biogenic amine production by lactic acid bacteria. International Journal of Food Microbiology, 53 (1): 33-41. Chamba, JF., Jamet, E. 2008: Contribution to the safety assessment of technological microflora found in fermented dairy products. International Journal of Food Microbiology, 126 (3): 263-266. Chiang, S-S., Pan, T-M. 2012: Beneficial effects of Lactobacillus paracasei subsp. paracasei NTU 101 and its fermented products. Applied Microbiology and Biotechnology, 93 (3): 903- 916. Danielsen, M., Wind, A. 2003: Susceptibility of Lactobacillus spp. to antimicrobial agents. International Journal of Food Microbiology, 82 (1): 1-11. De Vuyst, L., Leroy, F. 2007: Bacteriocins from lactic acid bacteria: production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology, 13 (4): 194- 199. Devirgiliis, C., Barile, S., Caravelli, A., Coppola, D., Perozzi, G. 2010: Identification of tetracycline‐ and erythromycin‐resistant Gram‐positive cocci within the fermenting microflora of an Italian dairy food product. Journal of Applied Microbiology, 109 (1): 313-323. Duary, RK., Rajput, YS., Batish, VK., Grover, S. 2011: Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells. The Indian Journal of Medical Research, 134 (5): 664-671. Dunne, C., O'Mahony, L., Murphy, L., Thornton, G., Morrissey, D., O'Halloran, S., Feeney, M., Flynn, S., Fitzgerald, G., Daly, C. 2001: In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. The American Journal of Clinical Nutrition, 73 (2): 386s-392s. FAO/WHO, 2002: Guidelines for the evaluation of probiotics in food. Report of a Joint FAO/WHO Working Groupon Drafting Guidelines for the Evaluation of Probiotics in Food.Ontario, Canada, April 30 and May 1, 2002, 1–11. Flórez, AB., Delgado, S., Mayo, B. 2005: Antimicrobial susceptibility of lactic acid bacteria isolated from a cheese environment. Canadian Journal of Microbiology, 51 (1): 51-58. Fortina, M., Ricci, G., Borgo, F., Manachini, P., Arends, K., Schiwon, K., Abajy, M., Grohmann, E. 2008: A survey on biotechnological potential and safety of the novel Enterococcus species of dairy origin, E. italicus. International Journal of Food Microbiology, 123 (3): 204-211. Franciosi, E., Settanni, L., Cavazza, A., Poznanski, E. 2009: Biodiversity and technological potential of wild lactic acid bacteria from raw cows' milk. International Dairy Journal, 19 (1): 3-11. Gajic, O., Kojic, M., Banina, A., Topisirovic, L. 1999: Characterization of natural isolate Lactococcus lactis subsp. lactis BGMN1-5, a strain producing two bacteriocins, cell wall- associated proteinase and showing clumping phenotype. Archives of Biological Sciences, 51: 69-78. Giaouris, E., Chapot-Chartier, M-P., Briandet, R. 2009: Surface physicochemical analysis of natural Lactococcus lactis strains reveals the existence of hydrophobic and low charged strains with altered adhesive properties. International Journal of Food Microbiology, 131 (1): 2-9. Iyer, R., Tomar, S., Kapila, S., Mani, J., Singh, R. 2010: Probiotic properties of folate producing Streptococcus thermophilus strains. Food Research International, 43 (1): 103-110. Janković, T., Frece, J., Abram, M., Gobin, I. 2012: Aggregation ability of potential probiotic Lactobacillus plantarum strains. International Journal of Sanitary Engineering Research, 6 (1): 17-22. Jokovic, N., Nikolic, M., Begovic, J., Jovcic, B., Savic, D., Topisirovic, L. 2008: A survey of the lactic acid bacteria isolated from Serbian artisanal dairy product kajmak. International Journal of Food Microbiology, 127 (3): 305-311. Joković, N., Rajković, J., Veljović, K., Tolinački, M., Topisirović, L. 2014: Screening of lactic acid bacteria isolated from Serbian kajmak for use in starter cultures. Biologica Nyssana, 5 (1): 37-46. Kastner, S., Perreten, V., Bleuler, H., Hugenschmidt, G., Lacroix, C., Meile, L. 2006: Antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food. Systematic and Applied Microbiology, 29 (2): 145-155. Katla, A-K., Kruse, H., Johnsen, G., Herikstad, H. 2001: Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. International Journal of Food Microbiology, 67 (1): 147-152. Kojic, M., Svircevic, J., Banina, A., Topisirovic, L. 1991: Bacteriocin-producing strain of Lactococcus lactis subsp. diacitilactis S50. Applied and Environmental Microbiology, 57 (6): 1835-1837. Kojic, M., Strahinic, I., Topisirovic, L. 2005: Proteinase PI and lactococcin A genes are located on the largest plasmid in Lactococcus lactis subsp. lactis bv. diacetylactis S50. Canadian Journal of Microbiology, 51 (4): 305-314. Landete, JM., de las Rivas, B., Marcobal, A., Muñoz, R. 2007: Molecular methods for the detection of BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 89 biogenic amine-producing bacteria on foods. International Journal of Food Microbiology, 117 (3): 258-269. Lozo, J., Vukasinovic, M., Strahinic, I., Topisirovic, A. 2004: Characterization and antimicrobial activity of bacteriocin 217 produced by natural isolate Lactobacillus paracasei subsp. paracasei BGBUK2-16. Journal of Food Protection, 67 (12): 2727-2734. Maldonado, NC., de Ruiz, CS., Otero, MC., Sesma, F., Nader-Macías, ME. 2012: Lactic acid bacteria isolated from young calves–characterization and potential as probiotics. Research in Veterinary Science, 92 (2): 342-349. Maragkoudakis, PA., Zoumpopoulou, G., Miaris, C., Kalantzopoulos, G., Pot, B., Tsakalidou, E. 2006: Probiotic potential of Lactobacillus strains isolated from dairy products. International Dairy Journal, 16 (3): 189-199. Martín-Platero, AM., Valdivia, E., Maqueda, M., Martínez-Bueno, M. 2009: Characterization and safety evaluation of enterococci isolated from Spanish goats' milk cheeses. International Journal of Food Microbiology, 132 (1): 24-32. Mathara, JM., Schillinger, U., Guigas, C., Franz, C., Kutima, PM., Mbugua, SK., Shin, H-K., Holzapfel, WH. 2008: Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya. International Journal of Food Microbiology, 126 (1): 57-64. Mohammed, M., El-Aziz, HA., Omran, N., Anwar, S., Awad, S., El-Soda, M. 2009: Rep-PCR characterization and biochemical selection of lactic acid bacteria isolated from the Delta area of Egypt. International Journal of Food Microbiology, 128 (3): 417-423. Nguyen, T., Kang, J., Lee, M. 2007: Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects. International Journal of Food Microbiology, 113 (3): 358-361. Pereira, DI., McCartney, AL., Gibson, GR. 2003: An in vitro study of the probiotic potential of a bile- salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties. Applied and Environmental Microbiology, 69 (8): 4743-4752. Pringsulaka, O., Rueangyotchanthana, K., Suwannasai, N., Watanapokasin, R., Amnueysit, P., Sunthornthummas, S., Sukkhum, S., Sarawaneeyaruk, S., Rangsiruji, A. 2015: In vitro screening of lactic acid bacteria for multi-strain probiotics. Livestock Science, 174 :66-73. Saavedra, L., Sesma, F., de Valdez, GF. 2003: Homemade traditional cheeses for the isolation of probiotic Enterococcus faecium strains. International Journal of Food Microbiology, 88 (2): 241-245. Stiles, ME., Holzapfel, WH. 1997: Lactic acid bacteria of foods and their current taxonomy. International Journal of Food Microbiology, 36 (1): 1-29. Tagg, J., McGiven, A. 1971: Assay system for bacteriocins. Applied Microbiology, 21 (5): 943. Temmerman, R., Pot, B., Huys, G., Swings, J. 2003: Identification and antibiotic susceptibility of bacterial isolates from probiotic products. International Journal of Food Microbiology, 81 (1): 1-10. Todorov, S., Botes, M., Guigas, C., Schillinger, U., Wiid, I., Wachsman, M., Holzapfel, W., Dicks, L. 2008: Boza, a natural source of probiotic lactic acid bacteria. Journal of Applied Microbiology, 104 (2): 465-477. Vujcic, M., Topisirovic, L. 1993: Molecular analysis of the rolling-circle replicating plasmid pA1 of Lactobacillus plantarum A112. Applied and Environmental Microbiology, 59 (1): 274-280. Xanthopoulos, V., Hatzikamari, M., Adamidis, T., Tsakalidou, E., Tzanetakis, N., Litopoulou- Tzanetaki, E. 2000: Heterogeneity of Lactobacillus plantarum isolates from Feta cheese throughout ripening. Journal of Applied Microbiology, 88 (6): 1056-1064. BIOLOGICA NYSSANA 6 (2)  December 2015: 81-89 Rajković, J., Joković, N.  Probiotic properties and safety assesment… 90